A conserved arginine with non-conserved function is a key determinant of agonist selectivity in α7 nicotinic ACh receptors

Background and Purpose The alpha 7 and alpha 4 beta 2* (* denotes possibly assembly with another subunit) nicotinic acetylcholine receptors (nAChRs) are the most abundant nAChRs in the mammalian brain. These receptors are the most targeted nAChRs in drug discovery programmes for brain disorders. However, the development of subtype-specific agonists remains challenging due to the high degree of sequence homology and conservation of function in nAChRs. We have developed C(10) variants of cytisine, a partial agonist of alpha 4 beta 2 nAChR that has been used for smoking cessation. The C(10) methyl analogue used in this study displays negligible affinity for alpha 7 nAChR, while retaining high affinity for alpha 4 beta 2 nAChR. Experimental Approach The structural underpinning of the selectivity of 10-methylcytisine for alpha 7 and alpha 4 beta 2 nAChRs was investigated using molecular dynamic simulations, mutagenesis and whole-cell and single-channel current recordings. Key Results We identified a conserved arginine in the beta 3 strand that exhibits a non-conserved function in nAChRs. In alpha 4 beta 2 nAChR, the arginine forms a salt bridge with an aspartate residue in loop B that is necessary for receptor expression, whereas in alpha 7 nAChR, this residue is not stabilised by electrostatic interactions, making its side chain highly mobile. This lack of constrain produces steric clashes with agonists and affects the dynamics of residues involved in agonist binding and the coupling network. Conclusion and Implications We conclude that the high mobility of the beta 3-strand arginine in the alpha 7 nAChR influences agonist binding and possibly gating network and desensitisation. The findings have implications for rational design of subtype-selective nAChR agents.

Automated summary

The alpha 7 and alpha 4 beta 2* nicotinic acetylcholine receptors are the most abundant nAChRs in the mammalian brain and are targeted in drug discovery for brain disorders. A C(10) variant of cytisine has been developed as a partial agonist of alpha 4 beta 2 nAChR. Molecular dynamic simulations and experiments revealed the structural basis for the selectivity of 10-methylcytisine for alpha 7 and alpha 4 beta 2 nAChRs, providing insights into agonist binding and receptor dynamics.